2.BIOCHEMISTRY DIVISION
Exploration of Susceptibility Genes for Colon Carcinogenesis by PhIP
2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) induces aberrant crypt foci (ACF) and colon tumors in rats. Two congenic rat strains, harboring the F344-derived colon cancer susceptibility gene (Sct) on rat chromosome 16, showed a higher susceptibility to PhIP-induced colon carcinogenesis than the ACI strain (49). Global gene expression analysis using a high-density oligonucleotide microarray revealed that more than 200 out of 8,740 genes were differentially expressed between F344 and ACI rats, the former being susceptible to colon carcinogenesis and the latter resistant. N-acetyltransferase 2 gene, Nat2, was highly expressed in F344 and two congenic rat strains, but was not localized right on the Sct region on chromosome 16. It is therefore conceivable that Nat2 may contribute to susceptibility to PhIP-induced colon carcinogenesis as one of the target genes of Sct. Nucleotide polymorphisms in the promoter sequence of Nat2 need to be investigated to elucidate the differential expression mechanism between F344 and ACI strains.
Multistep Genetic Alterations in Colon Carcinogenesis Induced by PhIP
Global gene expression analysis revealed that genes encoding Paneth cell-specific proteins, such as defensin family genes, lysozyme and MMP-7, were highly expressed in PhIP-induced rat colon tumors. Immunohistochemical analysis using an anti-lysozyme antibody demonstrated that Paneth cells were present even in high-grade dysplastic ACF, indicating that Paneth cell metaplasia of cryptic cells could be an early histomorphological change during PhIP-induced colon carcinogenesis. Some of the dysplastic ACF harbored genetic alterations in Apc or b-catenin gene, and mutation spectra in dysplastic ACF were quite similar to those observed in colon tumors (50). Dysplastic ACF were therefore considered to be preneoplastic lesions of the colon. Reports related to this work can be found in the list of publications (51-54).
Molecular Mechanisms for Maintenance of G-rich Short Tandem Repeats Stability
Minisatellite sequences (MNs) are frequently altered in various tumors (55). The tandem repeat of d(CAGGG)n in the mouse hypervariable MN Pc-1 was demonstrated to form an intramolecular quadruplex structure under physiological conditions and to cause arrest of DNA synthesis at the d(GGG)n site in vitro. Therefore, the characteristic quadruplex structure of Pc-1 and other Pc-1-like MN repeats could be responsible for the hypermutable feature of these MNs in vivo. UP1 has been identified as a single-stranded d(CAGGG)n binding protein. UP1 was demonstrated to unfold the intramolecular quadruplex structure of d(CAGGG)5 and to abrogate the arrest of DNA synthesis at d(GGG)n site (56). UP1 also unfolded unusual higher structures of telomeric d(TTAGGG)4 repeats and triplet d(CGG)n repeats, suggesting that UP1 is involved in the maintenance of genomic stability at G-rich short tandem repeats including telomeres in vivo.
Functional Analyses of DNA/RNA- Binding Protein, LRP130
LRP130 was originally isolated as a highly expressed protein with unknown functions in the human hepatoma HepG2 cell line. We identified a mouse homologue of LRP130 as a protein containing nine pentatricopeptide repeat motifs and binding to single-stranded cytosine-rich DNA sequences (57). LRP130 was also found to interact with mRNA in cells, and transient overexpression of full-length or the N-terminal region of LRP130 resulted in nuclear accumulation of poly(A)+ RNA in HeLa cells. These results suggest that LRP130 is involved in control of nucleo-cytoplasmic transport of mRNA, possibly by interacting with nuclear pore complexes through its N-terminal region.
Role of Poly(ADP-ribose) Metabolism in Carcinogenesis
Parp-1 is activated by DNA strand breaks and polyADP-ribosylates various chromatin-associated proteins, including Parp-1 itself (58,59). In Parp-1-/- mice, susceptibility to carcinogenesis induced by two alkylating agents, N-nitrosobis(2-hydroxypropyl) amine (BHP) and azoxymethane, significantly increased compared to the wild-type mice. Parp-1-/- mice harboring two marker genes, gpt and red/gam, for in vivo mutation analysis, were generated by crossbreeding with gpt delta transgenic mice. Deletion-type mutations and point mutations could be detected in red/gam and gpt genes, respectively. Parp-1-/- mice showed a higher frequency of red/gam gene mutants after BHP treatment. In contrast, mutant frequency in the gpt gene was not affected. It is therefore suggested that dysfunction of Parp-1 could not be fully compensated by other Parp family proteins or any other proteins involved in the DNA repair machinery for preventing deletion-type mutations. Parp-1 is also considered to be involved in transcriptional control. Induction of trophoblast giant cells and spongiotrophoblasts was observed during the development of teratocarcinoma-like tumors from Parp-1-/- ES cells grafted in nude mice (60). Parp-1-/- ES cells in culture also showed induction of trophoblast specific markers, including proliferin, Prlpa, and Tcfap2.
Poly(ADP-ribose) glycohydrolase (Parg) is a main enzyme, which degrades poly (ADP-ribose) into ADP-ribose. Since polyADP-ribosylated Parp-1 shows a marked decrease in PARP activity, inactivation of Parg may perturb the reversal of polyADP-ribosylated Parp-1 to become an active form. Cyclic peptide antibiotics, including pargamicin and PD124,966 were found to inhibit Parg activity (61). When the Parg gene was disrupted in mouse ES cells, Parg-/- ES cells showed increased lethality against treatment with alkylating agents and g-irradiation, suggesting that Parg is also involved in recovery from DNA damages.